The measurement of concentrations of such ligands as thyroid hormones in human blood serum is widely accepted as an important diagnostic aid in identifying particular functional disorders. For example, knowledge of the concentrations of thyroxine (T.sub.4) and triiodothyronine (T.sub.3) in human serum is of great usefulness to a diagnostician. Other ligands which lend themselves to quantitative analysis via radioimmunoassay include insulin, digoxin, and human growth hormone, and reference is made to the rather exhaustive treatise by Dr. Hans Georg Eckert reported in Angewandte Chemie, Volume 15, No. 9, September, 1976 at pages 525-533 for a non-exclusive list of ligands for which radioimmunoassay processes are feasible. Analyses by radioimmunoassay are particularly valuable for ligands having concentrations in blood serum at the nanogram to picogram per ml levels.
Broadly speaking, the radioimmunoassay procedure involves the use of an antibody which is specific to the ligand to be measured, and also a radioisotopically labeled ligand to which the antibody is also specific. The ligand to be measured and the labeled ligand compete for antibody binding sites. Eventually a measurable state is reached at which the relative concentrations of labeled and unlabeled ligands complexed or bound to the antibody are dependent upon the relative concentrations of the labeled and unlabeled ligands available and competing for the antibody binding sites. Since the amount of labeled ligand is constant, the amount of antibody-complexed labeled ligand will vary inversely with the amount of unlabeled ligand available and competing for the antibody binding sites. Folowing a suitable separation procedure, the amount of labeled ligand which remains bound to the antibody is indicated by radioisotope counting procedures (e.g., through the use of a gamma counter). The thus-indicated concentration of labeled ligand is compared with results from a number of control analyses involving samples having known concentrations of the ligand to be determined to infer the concentration of the unlabeled ligand.
Simplicity, accuracy, and reproducibility of radioimmunoassays or other assays involving competitive binding of labeled and unlabeled ligands to antibodies are highly desired features which contribute to the usefulness of such analyses in medical laboratories. It is evident that accuracy ordinarily decreases and the chances for error increase with the number of separate steps which must be taken in analyses of this type. To this end, it is preferable that few reagents (ideally, but a single reagent) be employed in such analyses, and that the time required to reach the desired degree of equilibration between the labeled and unlabeled ligand complexed with an antibody be as short as possible. Since, with some exceptions, equilibrium is approached more rapidly at increased temperatures, it is desired that the reagent or reagents be stable at relatively high temperatures. Further, it is necessary to isolate or separate the ligand-complexed antibody from the uncomplexed ligand during the assay procedure. It is important from the standpoint of accuracy that the separation procedure be carried out completely and reproducibly. When separation is performed by precipitation of the antibody-ligand complex, essentially none of the precipitate should be lost in the process, nor should the supernatant remain with the precipitate in any significant quantity.
Typical radioimmunoassay methods for thyroxine and triiodothyronine are taught in U.S. Pat. Nos. 3,911,096 and 3,928,553. The former patent describes a radioimmunoassay procedure for, for example, T.sub.4, which involves first incubating, for an hour, human serum to be tested in the presence of an anti-T.sub.4 antibody, radioisotopically labeled T.sub.4, and a "blocking agent" which prevents the binding of T.sub.4 to such blood proteins as thyroxine-binding globulin. The antibody, with its complexed T.sub.4 moieties, is then separated as, for example, by addition of a second antibody such as goat anti-rabbit gammaglobulin, which then causes precipitation. The latter patent contains similar teachings.